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ABSTRACT N -Homocysteinylation has been shown to induce immunogenic, thrombogenic, and amyloidogenic properties of proteins. Although very important to gain insight into the mechanisms of homocysteine (Hcy) toxicity, proteome-wide studies of the effects of Hcy-thiolactone (HTL) protein modification remain challenging due to the low abundance of N -Hcy-proteins. High field asymmetric waveform ion mobility spectrometry (FAIMS) has been shown to improve the identification of other PTMs, we therefore expected it to facilitate the characterization of N -homocysteinylated proteins ( N -Hcy-proteins) and help gain insight into their role in human disease. After extensive measurement optimization, we compared the yield of N -Hcy-protein/peptide identification across mouse liver and brain samples, either native or modified in vitro with HTL. Additionally, we examined the influence of different reduction and alkylation agents, namely DTT/IAA and TCEP/MMTS, on the number of identified N -Hcy-sites. FAIMS increased the number of N -Hcy-Lys-peptides and N -Hcy-proteins by 1.3-7-fold and 1.1-14-fold, respectively, regardless of alkylation method. We have identified 69 and 1,198 in vivo and in vitro N -Hcy-proteins, respectively. KEGG pathway term enrichment analysis showed that among in vitro N -Hcy-proteins, ten top KEGG pathways were Parkinson disease, prion disease, Huntington disease, oxidative phosphorylation, amyotrophic lateral sclerosis, pathways of neurodegeneration – multiple diseases, carbon metabolism, carcinogenesis – reactive oxygen species, Alzheimer disease, and diabetic cardiomyopathy. We conclude that FAIMS is a valuable addition to N -Hcy-proteome analysis workflow and facilitates the mapping of N -Hcy-sites. Data are available via ProteomeXchange with identifier PXD062860.
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